Although the rapid thrombin-induced release of arachidonic acid in human platelets has been known for over 20 years, the amount of arachidonic acid mass mobilized and the source of the released arachidonic acid has remained a subject of intense controversy. Herein, we exploit the analytic power and sensitivity of electrospray ionization mass spectrometry to identify plasmenylethanolamines as the largest source of arachidonic acid mass released during thrombin stimulation and to demonstrate the presence of multiple novel molecular species of plasmenylethanolamines in human platelets. Specifically, 90 s after thrombin stimulation a total of 60.1 nmol of arachidonic acid-containing phospholipids(109 platelets was hydrolyzed which included the loss of 3l.8 nmol/l0 platelets from ethanolamine glycerophospholipids (hydrolysis of plasmenylethanolamines represented 63% of the mass lost from the ethanolamine glycerophospholipid pool) but only 10.9 nmol/l09 platelets from choline glycerophospholipids. Human platelet phosphatidylserine and phosphatidylinositol pools contained similar amounts of arachidonic acid mass in resting platelets (-20 nmo1/l09 platelets), and each pool contributed 8.7 nmol/109 platelets after thrombin stimulation. From these results, a lower boundary for the rate of thrombin-induced arachidonic acid mobilization in human platelets can be set at -60 nmol/l09 platelets, thereby identifying specific kinetic characteristics and substrate selectivities of the phospholipase(s) activated during platelet stimulation. Collectively, these results underscore the importance of plasmenylethanolarmines as e major storage depot of arachidonic acid in resting platelets and as the major source of arachidonic acid mobilized after thrombin stimulation of human platelets.